JP2023533565A - ANTI-HUMAN IMMUNODEFICIENCE VIRUS-1 ANTIBODY AND METHOD OF USE THEREOF - Google Patents

Abstract

An anti-HIV-1 antibody comprising L-CDR1, L-CDR2 and L-CDR3, wherein L-CDR1 is SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, and any of SEQ ID NO: 15, 18 or 21 selected from the group consisting of sequences that differ by one or two substitutions, deletions or additions, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 16, 19 or 22; selected from the group consisting of sequences that differ by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 17, 20 or An antibody selected from the group consisting of any of the 23 and sequences that differ by one or two substitutions, deletions or additions.

Description

The present application relates to antibodies against human immunodeficiency virus-1 (anti-HIV-1) that specifically bind to the HIV-1 p24 protein. The invention also refers to methods and assays for detecting HIV-1 in samples using said antibodies.

Human Immunodeficiency Virus 1 (HIV-1) is a retrovirus that infects 37.9 million people worldwide and kills approximately 1 million people each year, especially in vulnerable populations unable to access diagnosis and treatment. (Soliman, M. et al., "Mechanisms of HIV Control," Current HIV/AIDS Reports 2017, vol. 14(3); pp. 101-9). HIV-1 is the major cause of acquired immunodeficiency syndrome (AIDS), a fatal disease transmitted from HIV-1 infected persons by sexual contact or by exposure to contaminated blood or blood-derived products. Viruses target the immune system by destroying and impairing immune cell function. Infected individuals become immunocompromised and susceptible to other opportunistic infections, as well as some types of cancer (Source from WHO website - https://www.who.int/news-room/fact-sheets/detail/ hiv-aids). Currently, only 46% of people living with HIV-1 know their infection status. Therefore, detection of HIV-1 during acute infection is a major public health concern (Stone, M. et al., Comparison of detection limits of fourth- and fifth-generation combination HIV antigen-antibody, p24 antigen , and viral load assays on diverse HIV isolates.”, Journal of Clinical Microbiology 2018, vol. 56(8); pp. 1-12).

In this particular situation, the goal is to diagnose HIV-1 within the first few weeks after the individual becomes infected (the acute phase), which is likely to prevent secondary infections and facilitate treatment and care. (Lewis J. et al., "Field accuracy of fourth-generation rapid diagnostic tests for acute HIV-1: a systematic review.", AIDS 2015, vol. 29(18);2465 ~71 pages). The use of early biomarkers for HIV-1 detection is key to achieving this goal in a timely manner. The most commonly used biomarkers for diagnosis of HIV-1 infection are antibodies against viral structural proteins. Here, p24 is the most abundant structural protein of the HIV-1 viral envelope and is secreted at high levels into the serum during the early stages of infection, making it an important biomarker for early HIV-1 detection. It is considered. p24 is a polymeric capsid protein that serves as the major structural component of the HIV-1 envelope around the viral RNA molecule. Like HIV-1 RNA, p24 is a 24-25 kDa protein derived from the Gag polyprotein precursor that is detectable prior to seroconversion (Gray, E.R. et al., p24 revisited: a landscape review of antigen detection). for early HIV diagnosis.”, AIDS. 2018, vol. 32(15); pp. 2089-102).

Current guidelines from the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend the use of fourth generation antibody-antigen assays as the preferred method for HIV-1 screening. These tests detected p24 antigen and anti-HIV-1 antibodies, narrowing the diagnostic gap from 4 weeks to 2 weeks after exposure (Gray, E.R., et al., p24 revisited: a landscape review of antigen detection for early HIV diagnosis.", AIDS. 2018, vol. 32(15); pp. 2089-102; Codoner, F. et al., "Gag protease coevolution analyzes define novel structural surfaces in the HIV-1 matrix and capsid involved in resistance to Protease Inhibitors .”, Scientific Reports 2017, vol. 7(3717); pp. 1-10; Alexander TS., “Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution.”, Clinical and Vaccine Immunology 2016, vol. 23(4); 249-53; WHO, "World Health Organization Model List of Essential In Vitro Diagnostics.", 1st edition, Geneva, 2018; Centers for Disease Control and Prevention, 2017, "National HIV testing day and new testing recommendations." Morbidity and Mortality Weekly Report vol.63(25);pp.537-37).

However, some antibodies currently on the market have low sensitivity for initial p24 detection, so there are still anti-HIV-1 antibodies that bind specifically to the p24 antigen, with high binding capacity and excellent manufacturing properties. It has been demanded.

Accordingly, the present invention provides anti-HIV-1 antibodies with improved binding capacity to the HIV-1 p24 protein compared to similar commercially available reagents. These antibodies recognize novel non-cross-reactive epitopes and can be used either alone or as capture/detection partners in multiple HIV-1 immunoassays, such as immunodiagnostic or blood screening platforms.

Soliman, M. et al., “Mechanisms of HIV Control,” Current HIV/AIDS Reports 2017, vol.14(3); pp.101-9 Source from WHO website - https://www.who.int/news-room/fact-sheets/detail/hiv-aids Stone, M. et al., “Comparison of detection limits of fourth- and fifth-generation combination HIV antigen-antibody, p24 antigen, and viral load assays on diverse HIV isolates.” Journal of Clinical Microbiology 2018, vol. 56(8) ; pages 1-12 Lewis J. et al., "Field accuracy of fourth-generation rapid diagnostic tests for acute HIV-1: a systematic review." AIDS 2015, vol. 29(18); pp. 2465-71. Gray, E.R. et al., "p24 revisited: a landscape review of antigen detection for early HIV diagnosis." AIDS. 2018, vol. 32(15); pp. 2089-102. Codoner, F. et al., "Gag protease coevolution analyzes define novel structural surfaces in the HIV-1 matrix and capsid involved in resistance to Protease Inhibitors." Scientific Reports 2017, vol. 7(3717); pp. 1-10 Alexander TS., "Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution." Clinical and Vaccine Immunology 2016, vol. 23(4); pp. 249-53 WHO, "World Health Organization Model List of Essential In Vitro Diagnostics.", 1st edition, Geneva, 2018; Centers for Disease Control and Prevention, 2017, "National HIV testing day and new testing recommendations.", Morbidity and Mortality Weekly Report vol.63(25);pp.537-37 Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. Chothia et al., Nature 342:877-883, 1989 MacCallum et al., J. Mol. Biol., 262:732-745, 1996. Lefranc, M.-P. Nucl. Acids Res., 33, pp. D593-D597, 2005 Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005) Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268. Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Altschul et al., 1990, J. Mol. Biol. 215:403-410. Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Lefranc M-P, Lefranc G, "IMGT® and 30 years of Immunoinformatics Insight in Antibody V and C Domain Structure and Function.", Jefferis R; Strohl W. R., Kato K., Antibodies 2019, vol. 8(29); 1-21 pages Liao-Chan S. et al., "Monoclonal Antibody Binding-site Diversity Assessment with a Cell-based Clustering Assay." Journal of Immunological Methods 2014, vol.405; pp. 1-14

When this specification refers to a sequence of CDR X or a sequence that differs from CDR X by one or two substitutions, deletions or additions, such substitutions, deletions or additions are amino acids defined by CDR X. It should be understood that any amino acid in the range of The present specification identifies each particular amino acid within the amino acid ranges defined by CDR X as suitable for such substitutions, deletions or additions.

As a non-limiting example, the L-CDR1 of Antibody #A can be defined as comprising the sequence of amino acids (1)-(11), RASQDISNYLH [shown schematically in SEQ ID NO: 15]. Each of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 may be substituted, deleted or added, unless otherwise specified or later refined by amendment. considered suitable.

In a first aspect, the present invention provides an anti-HIV-1 antibody comprising a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, and a sequence that differs from any of SEQ ID NO: 15, 18 or 21 by one or two substitutions, deletions or additions, wherein the amino acid sequence of L-CDR2 is L-CDR3 amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and a sequence that differs from any of SEQ ID NO: 16, 19 or 22 by one or two substitutions, deletions or additions discloses an antibody selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and sequences that differ by one or two substitutions, deletions or additions from any of SEQ ID NOs: 17, 20 or 23 do.

In other embodiments, an anti-HIV-1 antibody of the invention comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO:24, SEQ ID NO: 27, SEQ ID NO: 30, and sequences that differ by one or two substitutions, deletions or additions from any of SEQ ID NOs: 24, 27, or 30, wherein the amino acid sequence of H-CDR2 is the sequence the amino acid sequence of H-CDR3 selected from the group consisting of: No. 25, SEQ ID No. 28, SEQ ID No. 31, and a sequence that differs from any of SEQ ID Nos. 25, 28, or 31 by one or two substitutions, deletions or additions; is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, and sequences that differ from any of SEQ ID NOs: 26, 29, or 32 by one or two substitutions, deletions or additions.

In some embodiments, the light chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9. In other embodiments, the light chain comprises the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9.

In some embodiments, the heavy chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In other embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 15, or a sequence that differs from SEQ ID NO: 15 by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR2 comprises the sequence No. 16, or a sequence that differs from SEQ ID NO: 16 by one or two substitutions, deletions, or additions, wherein the amino acid sequence of L-CDR3 is SEQ ID NO: 17, or one or two substitutions, deletions from SEQ ID NO: 17 Includes sequences that differ due to deletions or additions. In other embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 18, or a sequence that differs from SEQ ID NO: 18 by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR2 comprises SEQ ID NO: 19, or a sequence that differs from SEQ ID NO: 19 by one or two substitutions, deletions, or additions, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20, or one or two substitutions, deletions from SEQ ID NO: 20 or contain additional sequences that differ. In other embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO:21 or a sequence that differs from SEQ ID NO:21 by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR2 comprises SEQ ID NO: 22, or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions, or additions, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23, or one or two substitutions, deletions from SEQ ID NO: 23 or contain additional sequences that differ.

In some embodiments, the amino acid sequence of H-CDR1 comprises SEQ ID NO:24, or a sequence that differs from SEQ ID NO:24 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR2 comprises the sequence No. 25, or a sequence that differs from SEQ ID No. 25 by one or two substitutions, deletions, or additions, and the amino acid sequence of H-CDR3 is SEQ ID No. 26, or one or two substitutions, deletions from SEQ ID No. 26. Includes sequences that differ due to deletions or additions. In other embodiments, the amino acid sequence of H-CDR1 comprises SEQ ID NO:27 or a sequence that differs from SEQ ID NO:27 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR2 comprises SEQ ID NO: 28, or a sequence that differs from SEQ ID NO: 28 by one or two substitutions, deletions, or additions, and the amino acid sequence of H-CDR3 is SEQ ID NO: 29, or one or two substitutions, deletions from SEQ ID NO: 29 or contain additional sequences that differ. In other embodiments, the amino acid sequence of H-CDR1 comprises SEQ ID NO:30 or a sequence that differs from SEQ ID NO:30 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR2 comprises SEQ ID NO: 31, or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions, or additions, and the amino acid sequence of H-CDR3 is SEQ ID NO: 32, or one or two substitutions, deletions from SEQ ID NO: 32 or contain additional sequences that differ.

In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 15, or a sequence that differs from SEQ ID NO: 15 by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR2 comprises the sequence No. 16, or a sequence that differs from SEQ ID NO: 16 by one or two substitutions, deletions, or additions, wherein the amino acid sequence of L-CDR3 is SEQ ID NO: 17, or one or two substitutions, deletions from SEQ ID NO: 17 The amino acid sequence of H-CDR1 comprises SEQ ID NO: 24 or a sequence that differs from SEQ ID NO: 24 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR2 comprises , SEQ ID NO:25, or a sequence that differs from SEQ ID NO:25 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR3 is SEQ ID NO:26, or one or two substitutions from SEQ ID NO:26 , includes sequences that differ due to deletions or additions.

In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 18, or a sequence that differs from SEQ ID NO: 18 by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR2 comprises the sequence No. 19, or a sequence that differs from SEQ ID NO: 19 by one or two substitutions, deletions, or additions, wherein the amino acid sequence of L-CDR3 is SEQ ID NO: 20, or one or two substitutions, deletions from SEQ ID NO: 20 The amino acid sequence of H-CDR1 comprises SEQ ID NO: 27 or a sequence that differs from SEQ ID NO: 27 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR2 comprises , SEQ ID NO:28, or a sequence that differs from SEQ ID NO:28 by one or two substitutions, deletions or additions, wherein the amino acid sequence of H-CDR3 is SEQ ID NO:29, or one or two substitutions from SEQ ID NO:29 , includes sequences that differ due to deletions or additions.

In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO:21, or a sequence that differs from SEQ ID NO:21 by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR2 comprises the sequence No. 22, or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions, or additions, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23, or one or two substitutions, deletions from SEQ ID NO: 23. The amino acid sequence of H-CDR1 comprises SEQ ID NO: 30 or a sequence that differs from SEQ ID NO: 30 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR2 comprises , SEQ ID NO:31, or a sequence that differs from SEQ ID NO:31 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR3 is SEQ ID NO:32, or one or two substitutions from SEQ ID NO:32 , includes sequences that differ due to deletions or additions.

In some embodiments, the light chain of an anti-HIV-1 antibody of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In some embodiments, the heavy chain of an anti-HIV-1 antibody of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

In some embodiments, the anti-HIV-1 antibodies of the invention specifically bind to an epitope of the HIV-1 p24 protein comprising the amino acid sequence of SEQ ID NO:33.

In some embodiments, the amino acid sequence of L-CDR1 of an anti-HIV-1 antibody of the invention comprises SEQ ID NO:21 or a sequence that differs from SEQ ID NO:21 by one or two substitutions, deletions or additions, The amino acid sequence of L-CDR2 includes SEQ ID NO: 22 or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions or additions, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23 or SEQ ID NO: 23. and sequences that differ by one or two substitutions, deletions or additions.

In some embodiments, the H-CDR1 amino acid sequence of the anti-HIV-1 antibody of the invention comprises SEQ ID NO:30 or a sequence that differs from SEQ ID NO:30 by one or two substitutions, deletions or additions, The amino acid sequence of H-CDR2 comprises SEQ ID NO: 31 or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions or additions, and the amino acid sequence of H-CDR3 is SEQ ID NO: 32 or SEQ ID NO: 32. and sequences that differ by one or two substitutions, deletions or additions.

In some embodiments, the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the antibody heavy chain comprises SEQ ID NO: 4, sequence No. 5, or SEQ ID No. 6.

In some embodiments, the anti-HIV-1 antibodies of the invention are monoclonal antibodies or recombinant antibodies. In another embodiment, said antibody is an antibody fragment. When the anti-HIV-1 antibody is an antibody fragment, variable fragment (Fv), single chain Fv (scFv), bispecific antibody (sc(Fv)2), single chain antibody, single domain antibody, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), diabodies, anti-idiotypic (anti-Id) antibodies, affibodies, nanobodies and Selected from unibody.

In some embodiments, the anti-HIV-1 antibody comprises a constant region of the murine IgG1 class or the murine IgG2a class.

In some embodiments, the anti-HIV-1 antibody is attached to a solid support.

In some embodiments, the invention discloses cells comprising the anti-HIV-1 antibodies of the invention.

In another aspect, the invention discloses a nucleic acid comprising a nucleotide sequence encoding an anti-HIV-1 antibody, a promoter operably linked to the nucleotide sequence, and a selectable marker. A cell comprising said nucleic acid is also disclosed herein.

The present invention also discloses compositions comprising an anti-HIV-1 antibody as described herein and a solid support, wherein the anti-HIV-1 antibody is covalently or non-covalently attached to the solid support. . In some embodiments, solid supports comprise solid phases of particles, beads, membranes, surfaces, polypeptide chips, microtiter plates, or chromatography columns.

The present invention also discloses a kit for detecting the presence of HIV-1 in a sample, said kit comprising at least one anti-HIV-1 antibody according to the invention and a solid support, wherein said at least one Antibodies are covalently or non-covalently attached to a solid support.

Percent monomer by SE-UPLC analysis of antibody #A and SDS-PAGE of antibody #A single clones (lanes 1, 2 and 3 represent subclones run under reducing and non-reducing conditions, respectively). Percent monomer by SE-UPLC analysis of antibody #B and SDS-PAGE of antibody #B single clones (lanes 1, 2 and 3 represent subclones run under reducing and non-reducing conditions, respectively). Percent monomer by SE-UPLC analysis of antibody #D and SDS-PAGE of antibody #D single clones (lanes 1, 2 and 3 represent subclones run under reducing and non-reducing conditions, respectively). PDB predicted structures of antibodies #A, #B and #D (4A, 4B and 4D respectively). For antibody #A the PDB structure code 2XKN was used in the homology query, for antibodies B# and #D the codes 5OPY and 1F3D respectively. Sensorgrams of saturated antibody #A and competing #B and #D. Antibodies #B and #D add signal to #A, indicating that these antibodies do not compete for binding within the same epitope region. Sensorgrams of saturated antibody #B and competing #A and #D. Antibodies #A and #D add signal to #B, indicating that these antibodies do not compete for binding within the same epitope region. Sensorgrams of saturated antibody #D and competing #A and #B. Antibodies #A and #B add signal to #D, indicating that these antibodies do not compete for binding within the same epitope region. Sensorgrams of the association of antibodies #A, #B and #D to HIV-1 p24 in the absence of competing antibodies. Each antibody achieves its full binding signal (experimental control). Binding kinetics of antibodies #A, #B and #D and commercial mAb #1 to antigen HIV-1 p24 calculated by biolayer interferometry (BLI). Sensorgrams were performed at gradient concentrations from 0.1 to 33 nM and fitted with a 1:1 binding model to calculate ka (association rate constant), kd (dissociation rate constant), KD (equilibrium dissociation constant). Binding of antibodies #A, #B and #D and commercial mAb #2 to HIV-1 p24 capsid protein by indirect ELISA. The titration curve for each antibody starts at a concentration of 2 μg/mL and is then diluted 1:10 (left). Signal-to-noise data for an antibody concentration of 200 ng/mL are shown on the right, demonstrating the poor performance of commercial mAb #2 when compared to antibodies #A, #B and #D.

The following description is intended merely to exemplify various embodiments of the present disclosure. As such, the specific modifications discussed are not intended to be limiting. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the spirit or scope of the subject matter presented herein, and such equivalent embodiments are provided herein. It is understood that should be included in the specification.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" do not clearly dictate otherwise. as long as it contains more than one referent.

Throughout this specification, unless the context requires otherwise, the word "comprises," or variations such as "comprises," or "comprising," refer to the elements or integers referred to, or the elements. or groups of integers are meant to be inclusive, but not to the exclusion of any other element or integer or group of elements or integers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although exemplary methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Each embodiment in this specification can be applied to any other embodiment mutatis mutandis unless expressly stated otherwise.

The following terms shall be understood to have the following meanings, unless otherwise indicated.

As used herein, the term "nucleic acid" refers to any material composed of DNA or RNA. Nucleic acids can be produced synthetically or by living cells.

As used herein, a "nucleotide" is a nucleic acid subunit consisting of a phosphate group, a five-carbon sugar and a nitrogenous base. The five-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogues of such subunits.

As used herein, the term "polynucleotide" refers to a polymeric chain of nucleotides. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNAs or RNAs containing non-natural nucleotide analogs, non-natural internucleoside linkages, or both. including analogues of Nucleic acids can adopt any topological structure. By way of example, nucleic acids can be single stranded, double stranded, triple stranded, quadruplex, partially double stranded, branched, hairpinned, circular, or padlock structures.

As used herein, the term "protein" refers to large biomolecules or macromolecules composed of one or more chains of amino acid residues. Many proteins are enzymes that catalyze biochemical reactions and are crucial for metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscles and proteins in the cytoskeleton that form the scaffolding system that maintains cell shape. Other proteins are important in cell signaling, immune response, cell adhesion, and cell cycle. However, the protein may also be wholly artificial or recombinant, ie not naturally occurring in living systems.

As used herein, the term "polypeptide" refers to both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives and analogs thereof. Polypeptides can be monomeric or polymeric. A polypeptide may contain several different domains (peptides), each of which has one or more distinct activities.

As used herein, the term "recombinant" means that (1) it has been removed from its naturally occurring environment, and (2) the gene has been combined with all or part of a polynucleotide found in nature. Refers to a biomolecule, eg, a gene or protein, that is unrelated, (3) operably linked to a polynucleotide with which it is not naturally linked, or (4) does not occur in nature. The term "recombinant" refers to cloned DNA isolates, chemically synthesized polynucleotide analogues, or polynucleotide analogues synthesized in vivo by heterologous systems, as well as nucleic acids encoded by such It can be used in connection with protein and/or mRNA.

As used herein, the term "fusion protein" refers to a protein containing two or more amino acid sequences that are not coexistent in the naturally occurring protein. A fusion protein may comprise two or more amino acid sequences from the same or different organisms. The two or more amino acid sequences of the fusion protein are typically in-frame, do not contain stop codons between them, and are typically translated from the mRNA as part of the fusion protein.

When referring to proteins according to (3), the term "fusion protein" and the term "recombinant" may be used interchangeably herein.

As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and are used interchangeably in the present invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen-binding site that specifically binds an antigen. Thus, the term antibody includes not only whole antibody molecules, but also antibody fragments or derivatives.

In a native antibody, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. A light chain comprises two domains, a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and specificity for antigen. Light (CL) and heavy (CH) chain constant region domains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR) . The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of one light chain variable part and one heavy chain variable part. Antibody specificity resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are composed primarily of residues that are from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the binding site. Complementarity Determining Regions or CDRs refer to amino acid sequences that collectively define the binding affinity and specificity of the natural Fv region of the native immunoglobulin binding site. Immunoglobulin light and heavy chains each have three CDRs, which are named L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. ing. An antigen-binding site therefore typically comprises six CDRs, with a set of CDRs from each of the heavy and light chain V-regions. Framework regions (FR) refer to amino acid sequences interposed between the CDRs.

CDRs are identified according to the definitions of Kabat, Chothia, accumulation of both Kabat and Chothia, AbM, contact, IMGT unique numbering, and/or conformational definitions, or any method of CDR determination known in the art. be able to. Antibody CDRs can be identified as the hypervariable regions originally defined by Kabat et al. See, eg, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs can also be identified as loop structures in the structure originally described by Chothia et al. (see, eg, Chothia et al., Nature 342:877-883, 1989). Other approaches to CDR identification include the "AbM definition" (a compromise between Kabat and Chothia and derived using Oxford Molecular's AbM antibody modeling software (now Accelrys0)), CDRs based on observed antigen contacts. (as presented in MacCallum et al., J. Mol. Biol., 262:732-745, 1996) or the "IMGT unique numbering" (relying on the high conservation of the structure of the variable regions (Lefranc , M.-P. Nucl. Acids Res., 33, D593-D597, 2005))). In another approach, referred to herein as "conformational definition" of CDRs, CDR positions can be identified as residues that produce enthalpy contribution to antigen binding. See, eg, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Still other CDR boundary definitions do not strictly follow one of the above approaches, but still overlap at least a portion of the Kabat CDRs, provided that a particular residue or group of residues or even the entire CDR is antigenic. They may be shortened or lengthened given predictions or experimental findings that binding is not significantly affected. As used herein, CDRs refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing multiple CDRs, the CDRs are Kabat numbering, Chothia numbering, extended numbering, AbM numbering, contact numbering, IMGT specific numbering, and/or or according to any of the conformational definitions.

An exemplary database of antibody sequences is the "Abysis" website at www.bioinf.org.uk/abs (maintained by A. C. Martin, Department of Biochemistry & Molecular Biology University College London, London, England) and Retter et al. , Nucl. Acids Res., 33 (Database issue): D671-D674 (2005), and can be accessed through the VBASE2 website at www.vbase2.org. Preferably, sequences are analyzed using Kabat, IMGT, and the Abysis database, which integrates sequence data from the Protein Data Bank (PDB) with structural data from the PDB. Unless otherwise indicated, all CDRs presented herein are derived according to the scheme shown according to the Abysis database website.

As used herein, the term "antibody" includes isolated antibodies, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies, recombinant Including antibodies, chimeric antibodies, and antibody fragments.

As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody composition having a homogeneous population of antibodies that bind the same epitope. The term is not limited with respect to the species or source of the antibody, nor is it intended to be limited by the method by which it is made. Thus, the term includes antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using humans rather than murine hybridomas. The term also includes antibodies obtained by other methods for producing monoclonal antibodies known in the art, such as establishment of eukaryotic cell lines by transient or stable transfection.

As used herein, the term "recombinant antibody" refers to an antibody expressed from a cell or cell line transfected with one or more expression vectors containing coding sequences for the antibody, wherein the coding sequences are Not naturally associated with cells. Recombinant antibodies or fragments thereof are prepared, expressed, produced or isolated by any recombinant means, as is well known to those of skill in the art.

In some embodiments, a "recombinant antibody" can also be a "monoclonal antibody" when it is derived from a homogeneous antibody population that binds the same epitope.

Thus, the term "antibody fragment" as used herein includes variable fragment (Fv), single chain Fv (scFv), bispecific antibody (sc(Fv) ₂ ), single chain antibody, single single domain antibodies, Fab fragments, F(ab') ₂ fragments, Fab' fragments, disulfide-linked Fvs (dsFv), chemically conjugated Fvs (ccFv), diabodies and anti-idiotypic (anti-Id) antibodies, and It includes, but is not limited to, functionally active epitope-binding fragments of any of the above. In certain embodiments, antibodies also include affibodies, nanobodies, and unibodies. In certain embodiments, specific antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (eg IgG, IgE, IgM, IgD, IgA and IgY), class (eg IgG1, IgG2, IgG3, IgG4, IgAi and IgA2) or subclass.

As used herein, the term "antigen-binding fragment (Fab)" refers to an antibody fragment that contains one constant domain and one variable domain for each of the heavy and light chains. The variable domain contains the antigen binding site. Antibodies generally comprise a fragment crystallizable region (Fc) and two antigen binding fragments (Fab). The Fab fragment can be separated from the Fc region to produce two Fab fragments, which are either F(ab') ₂ fragments or dimeric fragment antigen binding fragments. Also known as

The term "isolated" refers to a protein (eg, antibody) or nucleic acid that is substantially free of other cellular material and/or chemicals. For example, where the isolated antibody is expressed by cells from a different species, eg, a human antibody expressed in mouse cells will be substantially free of other proteins from the different species. A protein is substantially free of naturally associated components (or components associated with the cellular expression system used to produce the antibody) by isolation, using protein purification techniques well known in the art. can be prevented.

As used herein, the term "antigen" refers to a biomolecule that specifically binds to its respective antibody. Antibodies from a diverse repertoire bind specific antigenic structures through their variable region interactions.

As used herein, the term "epitope" refers to the portion of an antigen that is specifically bound by an antibody. Thus, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.

A polypeptide is "immunologically reactive" with an antibody if it binds to the antibody through antibody recognition of a specific epitope contained within that polypeptide. Immunological reactivity is determined by antibody binding, more specifically by the kinetics of antibody binding and/or by competition in binding using known polypeptides containing the epitope to which the antibody is directed as a competitor. can be determined. Techniques for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.

As used herein, the term "sample" refers to any biological material obtained from a subject or patient. In one aspect, the sample may comprise blood, ascites, CSF, saliva, or urine. In other embodiments, the sample may comprise whole blood, plasma, serum, B cells enriched from a blood sample, and cultured cells (eg, B cells from a subject). Samples can also include biopsies or tissue samples, including neural tissue. In still other embodiments, the sample may comprise whole cells and/or cell lysates.

The sample may be treated to physically or mechanically disrupt tissue or cellular structures, thereby releasing intracellular components into solution, which is further subjected to analysis using standard methods. Enzymes, buffers, salts, detergents, etc. used to prepare the biological sample for use may also be included. Samples can also include processed samples, such as those obtained by applying or passing the sample through a filtration device, or after centrifugation, or by attachment to a medium, matrix, or support.

The terms "patient" or "individual" are used interchangeably herein and refer to the mammalian subject being diagnosed or treated, with human patients being preferred. Optionally, the methods of the invention are useful in experimental animals, in veterinary applications, and in the development of animal models of disease, including, but not limited to, rodents, including mice, rats, and hamsters; and primates. Used in

The term "vector" refers to a nucleic acid that can be used to introduce into a cell another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments may be ligated. Another type of vector is a viral vector (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), wherein additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome.

An "expression vector" is a type of vector capable of directing the expression of a selected polynucleotide. An "expressing cell" is a cell that contains an expression vector.

A nucleotide sequence is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression of the nucleotide sequence (eg, the level, timing, or location of expression). A "regulatory sequence" is a nucleic acid that affects expression (eg, level, timing, or location of expression) of an operably linked nucleic acid. A regulatory sequence may, for example, exert its effect directly on the regulated nucleic acid or through the action of one or more other molecules (eg, a polypeptide that binds the regulatory sequence and/or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements.

As used herein, the terms "diagnostic" or "diagnosed" mean identifying the presence or nature of a pathological condition or a patient predisposed to a disease. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive ("percent true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not affected and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of people without disease who test positive. Although no particular diagnostic method can provide a definitive diagnosis of a condition, it is sufficient if the method provides a diagnostically positive indication.

As used herein, the term "binding affinity" refers to the strength of interaction between an epitope of an antigen and the antigen-binding site of an antibody.

The present invention relates to novel antibodies specific for the detection of Human Immunodeficiency Virus 1 (HIV-1) p24 protein. These antibodies recognize novel, non-cross-reactive epitopes of the HIV-1 p24 protein and exhibit a high degree of affinity and sensitivity compared to other commercial products. Accordingly, the antibodies described herein can be utilized as diagnostic reagents, standards or positive controls in immunoassays for early HIV-1 detection. They can be used to detect any of the three major groups of HIV-1: group M (main lineage), group N (novel), and group O (unclassified).

The invention also relates to compositions and kits containing said anti-HIV-1 antibodies for detecting the presence of HIV-1 in a sample.

I. Anti-HIV-1 Antibodies As used herein, the terms "homology,""similarity," or "identity," in the context of two or more nucleic acid or polypeptide sequences, are for maximum correspondence. Refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned to . To determine percent homology/identity, the sequences are aligned for optimal comparison (e.g., a first amino acid sequence or nucleic acid sequence is aligned for optimal alignment with a second amino acid sequence or nucleic acid sequence). gaps may be introduced in the sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences to be compared are the same length, after gaps have been introduced in their sequences if necessary (e.g., the sequences to be compared are (excluding additional sequences that extend beyond). For a sequence comparison between two sequences, "corresponding" CDRs refer to CDRs at the same location in both sequences (eg, CDR-H1 of each sequence).

The determination of percent identity, percent similarity, or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized to compare two sequences is the Karlin algorithm, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

In one embodiment described herein, a recombinant antibody comprises a light chain and a heavy chain. In other embodiments described herein, a recombinant antibody comprises two light chains and two heavy chains. A light chain of a recombinant antibody of the invention may comprise two domains, a variable domain (VL) and a constant domain (CL). A heavy chain of a recombinant antibody of the invention may comprise four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH).

In some embodiments, the anti-HIV-1 antibodies of the invention are monoclonal antibodies. In other embodiments, the anti-HIV-1 antibodies of the invention are recombinant antibodies. In other embodiments, the anti-HIV-1 antibody is a recombinant monoclonal antibody according to the definition of the invention. In other embodiments, the anti-HIV-1 antibody is an isolated antibody.

In some embodiments, the anti-HIV-1 antibody is an antibody fragment. In a preferred embodiment, said antibody fragment is a variable fragment (Fv), a single chain Fv (scFv), a bispecific antibody (sc(Fv) ₂ ), a single chain antibody, a single domain antibody, a Fab fragment, from F(ab') ₂ fragments, Fab' fragments, disulfide-linked Fvs (dsFv), chemically conjugated Fvs (ccFv), diabodies, anti-idiotypic (anti-Id) antibodies, affibodies, nanobodies, and unibodies selected.

In one embodiment described herein, the anti-HIV-1 antibody comprises an Fc region and two Fab fragments. In other embodiments described herein, the anti-HIV-1 antibody is an antigen-binding fragment and does not contain an Fc region. In other embodiments described herein, the anti-HIV-1 antibody consists of one Fab fragment. In other embodiments described herein, the anti-HIV-1 antibody consists of two Fab fragments (F(ab) ₂ ).

In one embodiment described herein, the anti-HIV-1 antibody is of any type known to those of skill in the art (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or (eg IgG1, IgG2, IgG3, IgG4, IgAi and IgA2) or any known subclass.

In one embodiment described herein, the anti-HIV-1 antibody is of the IgG type. In preferred embodiments, the anti-HIV-1 antibody is of the IgG1, IgG2, IgG3, or IgG4 class. In another preferred embodiment, the anti-HIV-1 antibody is of the IgG1 or IgG2 class. In another preferred embodiment, the anti-HIV-1 antibody is of the IgG2a class.

The species of the constant regions of the antibodies of the invention may be human, mouse, rabbit, rat, hamster, guinea pig, goat, sheep, horse, chicken, or a chimera of any of the foregoing species, although the Species are not particularly limited. In some preferred embodiments, the anti-HIV antibodies of the invention comprise constant regions of the murine IgG1 class or the murine IgG2a class.

A. Light Chains In some embodiments described herein, an anti-HIV-1 antibody comprises a light chain that includes complementarity determining regions (CDRs). Said CDRs correspond to sequences identified according to any CDR definition approach known to those skilled in the art. In some preferred embodiments, the CDR regions correspond to sequences identified according to the Kabat numbering scheme. In other preferred embodiments, the CDR regions may correspond to sequences identified according to other numbering schemes or combinations of Kabat and other numbering schemes. For example, the CDR regions can correspond to sequences identified according to the Chothia numbering scheme.

In some embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, each of which has SEQ ID NO: 7, or SEQ ID NO:8, or SEQ ID NO:9. In some preferred embodiments, the sequence of L-CDR1 is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18 and SEQ ID NO:21. In some preferred embodiments, the sequence of L-CDR2 is selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19 and SEQ ID NO:22. In some preferred embodiments, the sequence of L-CDR3 is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:23. In other preferred embodiments, the sequence of L-CDR1 is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 21, and the sequence of L-CDR2 is SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 22. and the sequence of L-CDR3 is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:23.

In another embodiment described herein, the variable region of the light chain of the anti-HIV-1 antibody of the invention comprises the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9. In yet another embodiment, the variable region of the light chain of the recombinant antibody is about 70%, 75%, 80%, 85% relative to the amino acid sequence consisting of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. In some preferred embodiments, the light chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9.

In another embodiment described herein, the recombinant antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In other embodiments, the light chain of the recombinant antibody is about 70%, 75%, 80%, 85%, 90%, They may have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. In some preferred embodiments, the light chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

B. Heavy Chains In some embodiments described herein, the anti-HIV-1 antibodies comprise heavy chains that include complementarity determining regions (CDRs). Said CDRs correspond to sequences identified according to any CDR definition approach known to those skilled in the art. In some preferred embodiments, the CDR regions correspond to sequences identified according to the Kabat numbering scheme. In other preferred embodiments, the CDR regions may correspond to sequences identified according to other numbering schemes or combinations of Kabat and other numbering schemes. For example, the CDR regions can correspond to sequences identified according to the Chothia numbering scheme.

In some embodiments described herein, the anti-HIV-1 antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, each of which has SEQ ID NO: 10, or SEQ ID NO:11, or SEQ ID NO:12. In some preferred embodiments, the H-CDR1 sequence is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27 and SEQ ID NO:30. In some preferred embodiments, the H-CDR2 sequence is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:28 and SEQ ID NO:31. In some preferred embodiments, the H-CDR3 sequence is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:32. In some preferred embodiments, the H-CDR1 sequence is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27 and SEQ ID NO:30, and the H-CDR2 sequence is SEQ ID NO:25, SEQ ID NO:28 and SEQ ID NO:28 31 and the sequence of H-CDR3 is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:32.

In another embodiment described herein, the variable region of the heavy chain of the anti-HIV-1 antibody of the invention comprises the amino acid sequence of SEQ ID NO:10, or SEQ ID NO:11, or SEQ ID NO:12. In yet another embodiment, the variable region of the heavy chain of the recombinant antibody is about 70%, 75%, 80%, 85% relative to the amino acid sequence consisting of SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. In some preferred embodiments, the heavy chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO:10, or SEQ ID NO:11, or SEQ ID NO:12.

In another embodiment described herein, the recombinant antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In other embodiments, the heavy chain of the recombinant antibody is about 70%, 75%, 80%, 85%, 90%, 91%, They may have 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. In some preferred embodiments, the heavy chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

C. Exemplary Anti-HIV-1 Antibodies In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3. , the amino acid sequence of L-CDR1 is SEQ ID NO:15, the amino acid sequence of L-CDR2 is SEQ ID NO:16, and the amino acid sequence of L-CDR3 is SEQ ID NO:17.

In other embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is the sequence 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20.

In other embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is the sequence 21, the amino acid sequence of L-CDR2 is SEQ ID NO:22, and the amino acid sequence of L-CDR3 is SEQ ID NO:23.

In one embodiment described herein, the anti-HIV-1 antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 24, the amino acid sequence of H-CDR2 is SEQ ID NO:25, and the amino acid sequence of H-CDR3 is SEQ ID NO:26.

In other embodiments described herein, the anti-HIV-1 antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is the sequence 27, the amino acid sequence of H-CDR2 is SEQ ID NO:28, and the amino acid sequence of H-CDR3 is SEQ ID NO:29.

In other embodiments described herein, the anti-HIV-1 antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is the sequence 30, the amino acid sequence of H-CDR2 is SEQ ID NO:31, and the amino acid sequence of H-CDR3 is SEQ ID NO:32.

The anti-HIV-1 antibodies of the invention can comprise any combination of both the light and heavy chain CDR regions described herein.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17. The amino acid sequence of H-CDR1 is SEQ ID NO:24, the amino acid sequence of H-CDR2 is SEQ ID NO:25, and the amino acid sequence of H-CDR3 is SEQ ID NO:26.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17. The amino acid sequence of H-CDR1 is SEQ ID NO:27, the amino acid sequence of H-CDR2 is SEQ ID NO:28, and the amino acid sequence of H-CDR3 is SEQ ID NO:29.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17. The amino acid sequence of H-CDR1 is SEQ ID NO:30, the amino acid sequence of H-CDR2 is SEQ ID NO:31, and the amino acid sequence of H-CDR3 is SEQ ID NO:32.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20; The amino acid sequence of H-CDR1 is SEQ ID NO:24, the amino acid sequence of H-CDR2 is SEQ ID NO:25, and the amino acid sequence of H-CDR3 is SEQ ID NO:26.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20; The amino acid sequence of H-CDR1 is SEQ ID NO:27, the amino acid sequence of H-CDR2 is SEQ ID NO:28, and the amino acid sequence of H-CDR3 is SEQ ID NO:29.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20; The amino acid sequence of H-CDR1 is SEQ ID NO:30, the amino acid sequence of H-CDR2 is SEQ ID NO:31, and the amino acid sequence of H-CDR3 is SEQ ID NO:32.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 21, the amino acid sequence of L-CDR2 is SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23; The amino acid sequence of H-CDR1 is SEQ ID NO:24, the amino acid sequence of H-CDR2 is SEQ ID NO:25, and the amino acid sequence of H-CDR3 is SEQ ID NO:26.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 21, the amino acid sequence of L-CDR2 is SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23; The amino acid sequence of H-CDR1 is SEQ ID NO:27, the amino acid sequence of H-CDR2 is SEQ ID NO:28, and the amino acid sequence of H-CDR3 is SEQ ID NO:29.

In preferred embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 21, the amino acid sequence of L-CDR2 is SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23; The amino acid sequence of H-CDR1 is SEQ ID NO:30, the amino acid sequence of H-CDR2 is SEQ ID NO:31, and the amino acid sequence of H-CDR3 is SEQ ID NO:32.

In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; A heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:11, or SEQ ID NO:12.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain comprising the amino acid sequence of SEQ ID NO:11.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain comprising the amino acid sequence of SEQ ID NO:12.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:8 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:8 and a heavy chain comprising the amino acid sequence of SEQ ID NO:11.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:8 and a heavy chain comprising the amino acid sequence of SEQ ID NO:12.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:11.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:12.

In other preferred embodiments, the light chain of the anti-HIV-1 antibody is about 70%, 75%, 80%, 85%, 90% relative to the amino acid sequence consisting of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology, the heavy chain of an anti-HIV-1 antibody is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 %, 96%, 97%, 98%, 99% or more homology.

In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; A heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5 or SEQ ID NO:6.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of SEQ ID NO:5.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of SEQ ID NO:6.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain comprising the amino acid sequence of SEQ ID NO:5.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain comprising the amino acid sequence of SEQ ID NO:6.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:5.

In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:6.

In other preferred embodiments, the light chain of the anti-HIV-1 antibody is about 70%, 75%, 80%, 85%, 90% relative to the amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology, the heavy chain of an anti-HIV-1 antibody is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% for an amino acid sequence consisting of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 %, 96%, 97%, 98%, 99% or more homology.

In some preferred embodiments, the anti-HIV-1 antibodies of the invention specifically bind to the HIV-1 p24 protein. In some embodiments, the anti-HIV-1 antibodies of the invention bind to an epitope of the HIV-1 p24 protein. In some preferred embodiments, the anti-HIV-1 antibodies of the invention bind to linear epitopes of the HIV-1 p24 protein. In some preferred embodiments, the anti-HIV-1 antibody of the invention comprises at least 5 selected from the amino acid sequence of the HIV-1 p24 protein (SEQ ID NO:35) or a sequence having at least 90% homology with said sequence. Binds a linear epitope containing three consecutive amino acids. In other embodiments, the amino acid sequence of the HIV-1 p24 protein is set forth in SEQ ID NO:36.

In another preferred embodiment, the anti-HIV-1 antibody of the invention binds to an epitope of the HIV-1 p24 protein characterized by comprising the amino acid sequence of SEQ ID NO:33.

In a more preferred embodiment, the anti-HIV-1 antibody of the invention binds to an epitope of the HIV-1 p24 protein characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, said antibody comprising: a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21; The amino acid sequence of L-CDR2 is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20 and SEQ ID NO: 23. and further comprising a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27 and SEQ ID NO: 30 The H-CDR2 amino acid sequence is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 28 and SEQ ID NO: 31, and the H-CDR3 amino acid sequence consists of SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 32 selected from the group.

In some preferred embodiments, the anti-HIV-1 antibody of the invention binds to an epitope of the HIV-1 p24 protein characterized by comprising the amino acid sequence of SEQ ID NO:33, said antibody comprising a complementarity determining region A light chain comprising L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is The amino acid sequence is SEQ ID NO: 20 and further comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, the amino acid sequence of H-CDR1 is SEQ ID NO: 27, H The amino acid sequence of -CDR2 is SEQ ID NO:28 and the amino acid sequence of H-CDR3 is SEQ ID NO:29.

In some embodiments, the anti-HIV-1 antibodies of the invention are attached to a solid support.

D. Affinity Tags An anti-HIV-1 antibody according to the invention may comprise an affinity tag. Affinity tags are useful for purification. Exemplary affinity tags include polyhistidine, glutathione S-transferase (GST), chitin binding protein, maltose binding protein (MBP), streptavidin binding peptide (Strep tag), isopeptide bond formation, FLAG tag, V5 tag, Myc tag, HA tag, NE tag, AviTag, calmodulin tag, polyglutamic acid, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, biotin carboxyl carrier Protein, green fluorescent protein tags, HaloTag, Nus tags, and thioredoxin tags include, but are not particularly limited to, the choice of affinity tag. That said, the anti-HIV-1 antibody may, for example, be purified after use if the affinity tag is removed or if the anti-HIV-1 antibody is purified using a strategy that does not require an affinity tag. May lack an affinity tag. An exemplary affinity tag is polyhistidine, which typically comprises an amino acid sequence containing between 4 and 10 consecutive histidines.

The anti-HIV-1 antibodies of the invention may contain an affinity tag and may be purified using said affinity tag. Several methods of purification of anti-HIV-1 antibodies are available in the state of the art and are well known to those skilled in the art. Exemplary methods of purification for anti-HIV-1 antibodies, with or without affinity tags, include immobilized metal affinity chromatography (IMAC), protein A/G affinity, exchange chromatography (IEX or IEC), Additional use of tag and affinity chromatography techniques over hydrophobic interaction chromatography (HIC) and/or IMAC or protein A/G. The purification methods and tags utilized should not be considered limiting.

II. Nucleic Acids, Cloning Cells, and Expression Cells The present invention also relates to nucleic acids comprising nucleotide sequences encoding the anti-HIV-1 antibodies described herein. The nucleic acid may be an isolated nucleic acid. Nucleic acids can be DNA or RNA. A DNA comprising a nucleotide sequence encoding an anti-HIV-1 antibody described herein typically includes a promoter operably linked to the nucleotide sequence. The promoter is preferably capable of driving constitutive or inducible expression of the nucleotide sequence in the intended expressing cells. The nucleic acid may also contain a selectable marker useful for selecting cells containing the nucleic acid of interest. Useful selectable markers are well known to those of skill in the art. The exact nucleotide sequence of the nucleic acid is not critical, so long as the nucleotide sequence encodes the anti-HIV-1 antibodies described herein. Codons may be chosen, for example, to match the codon bias of the expression cell of interest (eg, mammalian cells such as human cells) and/or for convenience during cloning. The DNA may be, eg, a plasmid, which may include an origin of replication (eg, for replication of the plasmid in prokaryotic cells).

In one embodiment described herein, a nucleic acid comprises a nucleotide sequence encoding an anti-HIV-1 antibody of the invention, a promoter operably linked to said nucleotide sequence, and a selectable marker.

In some preferred embodiments, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42. In a more preferred embodiment, the anti-HIV-1 antibody light chain nucleic acid of the invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:41, and the anti-HIV-1 antibody of the invention comprises a nucleotide sequence selected from the group consisting of: The -1 antibody heavy chain nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:40, and SEQ ID NO:42.

In some embodiments, the anti-HIV-1 antibody light and heavy chain nucleic acids of the invention comprise the nucleotide sequence of SEQ ID NO:37 and SEQ ID NO:38, respectively. In other embodiments, the anti-HIV-1 antibody light and heavy chain nucleic acids of the invention comprise the nucleotide sequences of SEQ ID NO:39 and SEQ ID NO:40, respectively. In other embodiments, the anti-HIV-1 antibody light and heavy chain nucleic acids of the invention comprise the nucleotide sequences of SEQ ID NO:41 and SEQ ID NO:42, respectively.

Various aspects of the invention also relate to cells containing a nucleic acid comprising a nucleotide sequence encoding an anti-HIV-1 antibody as described herein. Cells can be expression cells or cloned cells. Nucleic acids are typically cloned in E. coli, although other cloning cells may be used.

Where the cell is an expression cell, the nucleic acid may be a chromosomal nucleic acid, i.e. where the nucleotide sequence is integrated into the chromosome, provided that the nucleic acid is then in the expression cell e.g. , extrachromosomal DNA or vectors such as plasmids, cosmids, phages and the like. The type of vector should not be considered limiting.

In one embodiment described herein, the cells are typically expression cells. The nature of the expressing cells is not particularly limited. Mammalian expression cells may allow for convenient folding, post-translational modification, and/or secretion of recombinant or oligomeric recombinant antibodies, although other eukaryotic or prokaryotic cells may be used as expression cells. . Exemplary expressing cells include CHO cell lines such as TunaCHO or ExpiCHO, Expi293, BHK, NS0, Sp2/0, COS, C127, HEK, HT-1080, PER.C6, HeLa, and Jurkat cells. Cells may also be selected for vector integration, more preferably for plasmid DNA integration.

The anti-HIV-1 antibodies of the present invention can be produced by appropriate transfection strategies of nucleic acids containing nucleotide sequences encoding anti-HIV-1 antibodies into mammalian cells. Those skilled in the art are aware of the various techniques (lipofection, electroporation, etc.) available for transfection of nucleic acids into the cell line of choice. Therefore, the choice of mammalian cell line and transfection strategy should not be considered limiting. Cell lines can also be selected for integration of plasmid DNA.

In one preferred embodiment described herein, the cell comprises an anti-HIV-1 antibody of the invention.

III. Compositions and Kits Various aspects of the invention relate to compositions comprising anti-HIV-1 antibodies as described herein.

In one embodiment described herein, a composition comprises an anti-HIV-1 antibody of the invention and a solid support.

In other embodiments, a composition comprises an anti-HIV-1 antibody of the invention and a solid support, wherein said anti-HIV-1 antibody is covalently or non-covalently bound to said solid support. there is As used herein, the term "non-covalently bound" is exemplified, for example, by interactions between a streptavidin-binding protein and streptavidin or between an antibody and its antigen. specific binding, such as between an antibody and its antigen, between a ligand and its receptor, or between an enzyme and its substrate.

In other embodiments, a composition comprises an anti-HIV-1 antibody of the invention and a solid support, wherein said anti-HIV-1 antibody is directly or indirectly bound to the solid support. As used herein, the term "direct" binding refers to direct conjugation of a molecule to a solid support, e.g., a gold-thiol interaction that binds the cysteine thiol of an anti-HIV-1 antibody to a gold surface. Point. As used herein, the term "indirect" binding includes specific binding of an anti-HIV-1 antibody to another molecule that is directly attached to a solid support, e.g. Antibodies can bind antibodies that are directly bound to a solid support, thereby indirectly binding the anti-HIV-1 antibody to the solid support. The term "indirect" binding means that (a) each interaction between a daisy chain of molecules is a specific or covalent interaction and (b) the terminal molecule of the daisy chain is directly bound to the solid support. irrespective of the number of molecules between the anti-HIV-1 antibody and the solid support, as long as

Solid supports can include solid phases of particles, beads, membranes, surfaces, polypeptide chips, microtiter plates, or chromatography columns. Preferably, the solid support may be latex beads.

The composition may comprise a plurality of beads or particles, each bead or particle of said plurality of beads or particles being directly or indirectly associated with at least one anti-HIV-1 antibody as described herein. physically connected. The composition may comprise a plurality of beads or particles, each bead or particle of said plurality of beads or particles covalently binding to at least one anti-HIV-1 antibody as described herein or Non-covalently bound.

Various aspects of embodiments relate to a kit for detecting the presence of HIV-1 in a sample, said kit comprising at least one anti-HIV-1 antibody and a solid support or composition described herein. Including things. In some embodiments, at least one antibody is covalently or non-covalently attached to a solid support.

Anti-HIV-1 antibodies, compositions and kits described herein can be used, for example, in assays for detecting the presence of HIV-1 in a sample or for measuring the concentration of HIV-1 in a sample. but they are not limited to said assays. The anti-HIV-1 antibodies, compositions and kits of the invention can also be used for the detection of HIV-1 alone or in combination with other antibodies for the detection of other pathogens such as multiplex assays and methods. .

In some preferred embodiments, the anti-HIV-1 antibodies of the invention are used in methods and assays in which other RNA viruses are also detected. In other embodiments, the anti-HIV-1 and other anti-HIV-2 antibodies are used in methods and assays for simultaneous detection of HIV-1 and HIV-2 in a sample. In a more preferred embodiment, said anti-HIV-1 and other anti-HIV-2 antibodies are used in methods and assays for the specific detection of HIV-1 p24 and HIV-2 p26 proteins in a sample. .

Also contemplated within the scope of the present invention is the use of multiple anti-HIV-1 antibodies described herein in methods and assays for detecting HIV-1 in a sample.

The invention is explained in more detail below with reference to exemplary embodiments, which do not constitute a limitation of the invention.

(Example 1)
Generation of Preferred Anti-HIV Antibodies and Stable Cell Lines As disclosed herein, certain combinations of light and heavy chains of the present invention have resulted in preferred antibodies:

The variable and constant regions of each antibody were cloned into bicistronic vectors and expressed in Chinese Hamster Ovary (CHO) cells. The manufacturing properties of each antibody were evaluated based on their ability to generate stable cell line clones and reproducible expression and purification of functional antibodies.

Pool Development Transfection: Expression of the three constructs containing the nucleotide sequences of antibodies #A, #B and #D (SEQ ID NOS:37-42) in bicistronic expression vectors containing the heavy and light chains of each antibody. generated. For antibody expression, CHO cells were electroporated with 200 μg of DNA to generate stable cell lines. After 24 hours, transfected cells were counted and placed under selective medium for stable integration of protein genes.

Generation of pools: Transfected cells are seeded at a cell density of 0.5 x ¹⁰⁶ cells/mL in selective medium in 250 mL shaker flasks with a working volume of 50 mL, at 37°C, 5% _CO2. incubated. During the selection process, cells were spun down and resuspended in fresh selection medium every 2-3 days until the pool regained its growth rate and viability. Cell cultures were monitored for growth via viable cell density (VCD), percent viability, and titer.

Production pool: A 1 liter production run was performed on the stable pool to assess VCD, titer and viability. Cells were scaled up to production medium in 3 L shake flasks (1 L working volume). Conditioned media supernatants taken from each stable pool production run were clarified by centrifugation and proteins were purified by affinity purification using protein A columns (Tables 2-4).

Cell line banking: Cells were grown to 2.5×10 ⁶ per mL. Viability was greater than 95% at the time of harvest for cell banking. Cells were then centrifuged and the cell pellet resuspended in CHO complete medium containing 7.5% dimethylsulfoxide (DMSO) (Sigma-Aldrich, D1435) to obtain a cell count of 15 x ¹⁰⁶ cells/mL per vial. and A total of 5 vials of each pool were made and cryopreserved for storage in liquid nitrogen.

Generation of Stable Antibodies Starting with the best banked pooled cell lines for antibodies #A, #B and #D, stable clones were obtained by single cell cloning. The best clone for each antibody was selected based on expression levels and bioanalytical characterization of purified material of antibodies #A, #B and #D from the manufacturing process. Bioanalytical characterization included SE-UPLC and SDS-PAGE (Figures 1-3).

(Example 2)
Antibody Modeling and Evaluation Three-dimensional structural models of antibodies #A, #B and #D were constructed by antibody homology using the computational modeling software Bioiluminate (Schrodinger), version 3.5. Briefly, the amino acid sequences of the VH and VL regions of antibodies #A, #B and #D were loaded into Bioiluminate. Framework regions and CDRs were identified by searching antibody structures in the protein data bank (PDB) and selecting PDB templates based on high sequence similarity and structural compatibility (Table 5). The predicted CDR sequences for each antibody of the invention are shown in Table 5, and the PDB predicted structures of antibodies #A, #B and #D are shown in Figures 4A, 4B and 4D respectively. For antibody #A the PDB structure code 2XKN was used in the homology query, for antibodies B# and #D the codes 5OPY and 1F3D respectively.

Antibodies #A and #B were of the IgG1k isotype and antibody #D was of the IgG2ak isotype.

Analysis of the nucleotide sequences of the three antibodies was queried against IgBLAST, an algorithm developed by the National Center for Biotechnology Information (NCBI) that facilitates analysis of immunoglobulin variable domain sequences against the ImMunoGeneTics Database (IMGT) database. It has been shown that all heavy (VH) and light (VL) chains generated have unique complementarity determining regions (CDRs) (Lefranc M-P, Lefranc G, IMGT Trademark) and 30 years of Immunoinformatics Insight in Antibody V and C Domain Structure and Function.”, Jefferis R; Strohl W. R., Kato K., Antibodies 2019, vol. 8(29); pp. 1-21).

(Example 3)
Epitope Mapping of mAb D To avoid truncated peptides, the sequence of HIV-p24 was extended by neutral GSGSGSG linkers at the C- and N-termini. The extended antigen sequence was translated into 15 amino acid linear peptides with a 14 amino acid peptide-peptide overlap. The resulting HIV-p24 peptide microarray contained 232 different linear peptides (464 spots) printed in duplicate, plus additional HA (YPYDVPDYAG, 38 spots) and c-Myc ( EQKLISEEDL, 38 spots) were shaped by the control peptide.

Wash buffer: PBS with 0.05% Tween 20, pH 7.4; 3 x 10 second washes after each incubation step

Blocking Buffer: Rockland Blocking Buffer MB-070 (30 minutes before first assay)

Incubation buffer: wash buffer containing 10% blocking buffer

Assay conditions: antibody concentrations of 1 μg/ml, 10 μg/ml and 100 μg/ml in incubation buffer; incubation for 16 h at 4° C.; shaking at 140 rpm.

Secondary antibody: goat anti-mouse IgG(H+L) DyLight680 (0.2 μg/ml); staining for 45 minutes in incubation buffer at RT

Control antibody: mouse monoclonal anti-HA (12CA5) DyLight800 (0.5 μg/ml); stained for 45 minutes in incubation buffer at RT

Scanner: LI-COR Odyssey Imaging System; scan offset 0.65 mm, resolution 21 μm, scan intensity 7/7 (red=680 nm/green=800 nm)

Pre-staining of HIV-p24 peptide microarrays was performed using a secondary goat anti-mouse IgG(H+L) DyLight680 antibody in incubation buffer to investigate background interactions with antigen-derived peptides that could interfere with the primary assay. I used it. Subsequent incubation of other HIV-p24 peptide microarray copies with monoclonal antibody D at concentrations of 1 μg/ml, 10 μg/ml and 100 μg/ml in incubation buffer followed by staining with secondary and control antibodies; Readings were taken at a scan intensity of /7 (red/green). Additional HA peptides forming the peptide microarray were then stained as an internal quality control to confirm assay quality and peptide microarray integrity.

Epitope mapping to HIV-p24 for mAb D and subsequent epitope replacement scan highlighted a conserved 7-amino acid core motif PIAPGQM (SEQ ID NO:33).

(Example 4)
Epitope binning studies Molecular docking and Western blot evaluation of antibodies #A, #B and #D suggested that these antibodies recognized linear epitopes in regions 1, 4 and 7 of the HIV-1 p24 protein, respectively. . To further confirm these observations, a tandem epitope binning assay was performed using biolayer interferometry (BLI). A yeast-derived version of the HIV-1 p24 antigen was biotinylated (bt-p24) and loaded onto the streptavidin (SA) biosensor for 300 seconds. The loaded sensor was soaked in saturating antibody (100 μg/mL) for 600 seconds followed by competing antibody (25 μg/mL) for 300 seconds. The results show that antibodies #B and #D add an increase in the BLI signal response when antibody #A binds to HIV-1 p24, which is similar to antibody # B and #D show that they bind to different epitopes. Similarly, when antibody #A or #D was used as a saturating antibody, the remaining antibodies show no competition for the same epitope (Figures 9-10).

Table 6 summarizes the epitope binning data for antibodies #A, #B and #D. Briefly, the BLI signals of competing and saturating antibodies were normalized to buffer. The threshold for determining antibody blocking or binding was set at 0.02 to allow recognition of self-blocking pairs on the diagonal of the matrix (grey indicates binding, bold indicates self-blocking). The PEARSON correlation coefficient for the first antibody #A was calculated using the PEARSON function in Microsoft Excel (Liao-Chan S. et al., "Monoclonal Antibody Binding-site Diversity Assessment with a Cell-based Clustering Assay." Journal of Immunological Methods 2014, vol.405; pp.1-14). Three different bins were identified for antibodies #A, #B and #D. No antibody blocking was observed.

(Example 5)
Affinity Assessment of Anti-HIV-1 Antibodies #A, #B and #D To investigate in more detail the interaction between antibodies #A, #B and #D and the HIV-1 p24 antigen, affinity assays were performed by BLI. A sex analysis was performed. Antibodies #A, #B and #D were compared to a commercially available monoclonal antibody (commercial mAb #1). Biosensor chips (ForteBio) specifically coated with anti-mouse Fc were used to capture antibodies #A, #B and #D, as well as commercial mAb #1. The concentration gradients used for each antibody ranged from 0.1 to 33 nM, and each dilution consisted of 0.01% (w/v) bovine serum albumin (BSA) and 0.02% (v/v) surfactant Tween-20. Prepared in acid buffer (PBS). The recorded sensorgrams were fitted using a 1:1 binding model, and the equilibrium constant KD was calculated from the ratio of dissociation rate to association rate (kd/ka). The tested antibodies were ranked based on their calculated affinity constants as follows: Antibody #B≈Antibody #D>Antibody #A>Commercial mAb #1. Due to the long dissociation curves observed, it was not possible to calculate exact KD values for antibodies #B and #D, but the data presented show the calculated KDs for antibodies #A, #B and #D. values are lower than those observed for commercial mAb #1 (Table 7). This data supports the observation that antibodies #A, #B and #D show higher affinity for HIV-1 p24 than commercial mAb #1.

(Example 6)
Binding capacity of anti-HIV-1 antibodies #A, #B and #D
To further evaluate the binding of antibodies #A, #B and #D to the HIV-1 p24 antigen, an indirect ELISA assay was performed. A titration curve for each antibody was generated using a starting concentration of 2 μg/mL and making 1:10 serial dilutions to reach concentrations as low as 2×10 ⁻² ng/mL. The performance of each antibody was compared to a commercial clone (commercial mAb #2) (Figure 10, left). The data show that antibodies #A, #B and #D bind with higher signal-to-noise ratios (S/N) than the commercial antibodies, primarily at concentrations of 20-2000 ng/mL, and that these are the commercial mAbs. It also exhibits lower EC50 values compared to #2 (Figure 10, right).

CONCLUSION A functional assay comparing anti-HIV-1 antibodies #A, #B and #D with commercial anti-HIV-1 p24 antibodies from commercial mAbs #1 and #2 was performed. The binding affinity and potency of each antibody were evaluated by BLI and indirect ELISA. In both experiments, HIV-1 antibodies #A, #B and #D show superior affinities and superior EC50 values than the commercial antibodies tested (see Figure 9 and Table for kinetic analysis). 7 (Table 7), Figure 10 for ELISA data).

The experimental data presented here demonstrate that the anti-HIV-1 antibodies of the invention can be used to detect the p24 structural protein of HIV-1. Said antibodies exhibit improved properties with respect to affinity, sensitivity, potency, expression, solubility and manufacturability when compared to similar products on the market, and their use in serological tests is It contributes to shortening the time window between HIV-1 infection and a diagnostic event, thus preventing secondary viral transmission.

Claims (32)

An anti-HIV-1 antibody comprising a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3,
The amino acid sequence of L-CDR1 is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, and a sequence that differs from any of SEQ ID NO: 15, 18 or 21 by one or two substitutions, deletions or additions. is,
The amino acid sequence of L-CDR2 is from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and a sequence that differs from any of SEQ ID NO: 16, 19, or 22 by one or two substitutions, deletions, or additions. selected,
The amino acid sequence of L-CDR3 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and a sequence that differs from any of SEQ ID NO: 17, 20 or 23 by one or two substitutions, deletions or additions. , anti-HIV-1 antibodies. the antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3;
The amino acid sequence of H-CDR1 is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, and a sequence that differs from any of SEQ ID NO: 24, 27 or 30 by one or two substitutions, deletions or additions. is,
The amino acid sequence of H-CDR2 is from the group consisting of SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, and a sequence that differs from any of SEQ ID NO: 25, 28, or 31 by one or two substitutions, deletions, or additions. selected,
The amino acid sequence of H-CDR3 is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, and a sequence that differs from any of SEQ ID NO: 26, 29 or 32 by one or two substitutions, deletions or additions. The anti-HIV-1 antibody of claim 1, wherein the antibody is 3. The anti-HIV-1 antibody of claim 1 or 2, wherein the light chain comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. 4. The anti-HIV-1 antibody of claim 3, wherein the light chain comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. 5. The anti-HIV-1 antibody of any one of claims 1-4, wherein the heavy chain comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. . 6. The anti-HIV-1 antibody of claim 5, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. the amino acid sequence of L-CDR1 comprises SEQ ID NO: 15 or a sequence that differs from SEQ ID NO: 15 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR2 comprises SEQ ID NO: 16 or a sequence that differs from SEQ ID NO: 16 by one or two substitutions, deletions or additions;
7. The anti-HIV-A according to any one of claims 1 to 6, wherein the amino acid sequence of L-CDR3 comprises SEQ ID NO: 17 or a sequence that differs from SEQ ID NO: 17 by one or two substitutions, deletions or additions. 1 antibody. the amino acid sequence of L-CDR1 comprises SEQ ID NO: 18 or a sequence that differs from SEQ ID NO: 18 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR2 comprises SEQ ID NO: 19 or a sequence that differs from SEQ ID NO: 19 by one or two substitutions, deletions or additions;
Anti-HIV-anti-HIV- according to any one of claims 1 to 6, wherein the amino acid sequence of L-CDR3 comprises SEQ ID NO: 20 or a sequence that differs from SEQ ID NO: 20 by one or two substitutions, deletions or additions. 1 antibody. the amino acid sequence of L-CDR1 comprises SEQ ID NO: 21 or a sequence that differs from SEQ ID NO: 21 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR2 comprises SEQ ID NO: 22 or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions or additions;
Anti-HIV-anti-HIV- according to any one of claims 1 to 6, wherein the amino acid sequence of L-CDR3 comprises SEQ ID NO: 23 or a sequence that differs from SEQ ID NO: 23 by one or two substitutions, deletions or additions. 1 antibody. the amino acid sequence of H-CDR1 comprises SEQ ID NO: 24 or a sequence that differs from SEQ ID NO: 24 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR2 comprises SEQ ID NO: 25 or a sequence that differs from SEQ ID NO: 25 by one or two substitutions, deletions or additions;
8. The anti-HIV-A according to any one of claims 1 to 7, wherein the amino acid sequence of H-CDR3 comprises SEQ ID NO: 26 or a sequence that differs from SEQ ID NO: 26 by one or two substitutions, deletions or additions. 1 antibody. the amino acid sequence of H-CDR1 comprises SEQ ID NO: 27 or a sequence that differs from SEQ ID NO: 27 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR2 comprises SEQ ID NO: 28 or a sequence that differs from SEQ ID NO: 28 by one or two substitutions, deletions or additions;
9. The anti-antibody according to any one of claims 1 to 6 or 8, wherein the amino acid sequence of H-CDR3 comprises SEQ ID NO: 29, or a sequence that differs from SEQ ID NO: 29 by one or two substitutions, deletions or additions. HIV-1 antibody. the amino acid sequence of H-CDR1 comprises SEQ ID NO: 30 or a sequence that differs from SEQ ID NO: 30 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR2 comprises SEQ ID NO: 31 or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions or additions;
10. The anti-antibody according to any one of claims 1 to 6 or 9, wherein the amino acid sequence of H-CDR3 comprises SEQ ID NO: 32, or a sequence that differs from SEQ ID NO: 32 by one or two substitutions, deletions or additions. HIV-1 antibody. the amino acid sequence of L-CDR1 comprises SEQ ID NO: 15 or a sequence that differs from SEQ ID NO: 15 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR2 comprises SEQ ID NO: 16 or a sequence that differs from SEQ ID NO: 16 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR3 comprises SEQ ID NO: 17 or a sequence that differs from SEQ ID NO: 17 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR1 comprises SEQ ID NO: 24 or a sequence that differs from SEQ ID NO: 24 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR2 comprises SEQ ID NO: 25 or a sequence that differs from SEQ ID NO: 25 by one or two substitutions, deletions or additions;
7. The anti-HIV-A according to any one of claims 1 to 6, wherein the amino acid sequence of H-CDR3 comprises SEQ ID NO: 26 or a sequence that differs from SEQ ID NO: 26 by one or two substitutions, deletions or additions. 1 antibody. the amino acid sequence of L-CDR1 comprises SEQ ID NO: 18 or a sequence that differs from SEQ ID NO: 18 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR2 comprises SEQ ID NO: 19 or a sequence that differs from SEQ ID NO: 19 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR3 comprises SEQ ID NO: 20 or a sequence that differs from SEQ ID NO: 20 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR1 comprises SEQ ID NO: 27 or a sequence that differs from SEQ ID NO: 27 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR2 comprises SEQ ID NO: 28 or a sequence that differs from SEQ ID NO: 28 by one or two substitutions, deletions or additions;
7. The anti-HIV-A according to any one of claims 1 to 6, wherein the amino acid sequence of H-CDR3 comprises SEQ ID NO: 29 or a sequence that differs from SEQ ID NO: 29 by one or two substitutions, deletions or additions. 1 antibody. the amino acid sequence of L-CDR1 comprises SEQ ID NO: 21 or a sequence that differs from SEQ ID NO: 21 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR2 comprises SEQ ID NO: 22 or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR3 comprises SEQ ID NO: 23 or a sequence that differs from SEQ ID NO: 23 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR1 comprises SEQ ID NO: 30 or a sequence that differs from SEQ ID NO: 30 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR2 comprises SEQ ID NO: 31 or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions or additions;
7. The anti-HIV-A according to any one of claims 1 to 6, wherein the amino acid sequence of H-CDR3 comprises SEQ ID NO: 32 or a sequence that differs from SEQ ID NO: 32 by one or two substitutions, deletions or additions. 1 antibody. 7. The anti-HIV-1 antibody of any one of claims 1-6, wherein the light chain of said antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. 7. The anti-HIV-1 antibody of any one of claims 1-6, wherein the heavy chain of said antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the antibody heavy chain comprises SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 7. The anti-HIV-1 antibody of any one of claims 1-6, comprising an amino acid sequence selected from the group consisting of: 19. The anti-HIV-1 antibody according to any one of claims 1-18, which is a monoclonal antibody or a recombinant antibody. 20. The anti-HIV-1 antibody of any one of claims 1-19, which is an antibody fragment. Antibody fragments include variable fragment (Fv), single chain Fv (scFv), bispecific antibody (sc(Fv) ₂ ), single chain antibody, single domain antibody, Fab fragment, F(ab') ₂ Fragments, Fab' fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), diabodies, anti-idiotypic (anti-Id) antibodies, affibodies, nanobodies, and unibodies. 20. The anti-HIV-1 antibody according to 20. The anti-HIV-1 antibody according to any one of claims 1 to 21, comprising a constant region of mouse IgG1 class or mouse IgG2a class. 23. The anti-HIV-1 antibody of any one of claims 1-22, bound to a solid support. A cell comprising an anti-HIV-1 antibody according to any one of claims 1-23. A nucleic acid comprising a nucleotide sequence encoding the anti-HIV-1 antibody of any one of claims 1-20, a promoter operably linked to said nucleotide sequence, and a selectable marker. 26. A cell comprising the nucleic acid of claim 25. 21. The anti-HIV-1 antibody of any one of claims 1-20, wherein the anti-HIV-1 antibody is covalently or non-covalently bound to the solid support, and a solid support. Composition. 28. The composition of claim 27, wherein the solid support comprises the solid phase of a particle, bead, membrane, surface, polypeptide chip, microtiter plate, or chromatography column. A kit for detecting the presence of HIV-1 in a sample, said kit comprising at least one anti-HIV-1 antibody according to any one of claims 1-23, and a solid support. , wherein said at least one anti-HIV-1 antibody is covalently or non-covalently attached to a solid support. An anti-HIV-1 antibody that specifically binds to an epitope of the HIV-1 p24 protein comprising the amino acid sequence of SEQ ID NO:33. the amino acid sequence of L-CDR1 comprises SEQ ID NO: 21 or a sequence that differs from SEQ ID NO: 21 by one or two substitutions, deletions or additions;
the amino acid sequence of L-CDR2 comprises SEQ ID NO: 22 or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions or additions;
31. The anti-HIV-1 antibody of claim 30, wherein the amino acid sequence of L-CDR3 comprises SEQ ID NO:23 or a sequence that differs from SEQ ID NO:23 by one or two substitutions, deletions or additions. the amino acid sequence of H-CDR1 comprises SEQ ID NO: 30 or a sequence that differs from SEQ ID NO: 30 by one or two substitutions, deletions or additions;
the amino acid sequence of H-CDR2 comprises SEQ ID NO: 31 or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions or additions;
32. Anti-HIV-A according to any one of claims 30 to 31, wherein the amino acid sequence of H-CDR3 comprises SEQ ID NO: 32 or a sequence that differs from SEQ ID NO: 32 by one or two substitutions, deletions or additions. 1 antibody.

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